Irina G. Rybalkina

PDE5 is converted to an activated state upon cGMP binding to the GAF A domain

cGMP‐specific, cGMP‐binding phosphodiesterase (PDE5) regulates such physiological processes as smooth muscle relaxation and neuronal survival. PDE5 contains two N‐terminal domains (GAF A and GAF B), but the functional roles of these domains have not been determined. Here we show that recombinant PDE5 is activated directly upon cGMP binding to the GAF A domain, and this effect does not require PDE5 phosphorylation. PDE5 exhibited time‐ and concentration‐dependent reversible activation in response to cGMP, with the highest activation (9‐ to 11‐fold) observed at low substrate concentrations (0.1 μM cGMP). A monoclonal antibody directed against GAF A blocked cGMP binding, prevented PDE5 activation and decreased basal activity, revealing that PDE5 in its non‐activated state has low intrinsic catalytic activity. Activated PDE5 showed higher sensitivity towards sildenafil than non‐activated PDE5. The stimulatory effect of cGMP binding on the catalytic activity of PDE5 suggests that this mechanism of enzyme activation may be common among other GAF domain‐containing proteins. The data also suggest that development of agonists and antagonists of PDE5 activity based on binding to this site might be possible.

Calmodulin-stimulated cyclic nucleotide phosphodiesterase (PDE1C) is induced in human arterial smooth muscle cells of the synthetic, proliferative phenotype.

The diversity among cyclic nucleotide phosphodiesterases provides multiple mechanisms for regulation of cAMP and cGMP in the cardiovascular system. Here we report that a calmodulin-stimulated phosphodiesterase (PDE1C) is highly expressed in proliferating human arterial smooth muscle cells (SMCs) in primary culture, but not in the quiescent SMCs of intact human aorta. High levels of PDE1C were found in primary cultures of SMCs derived from explants of human newborn and adult aortas, and in SMCs cultured from severe atherosclerotic lesions. PDE1C was the major cAMP hydrolytic activity in these SMCs. PDE expression patterns in primary SMC cultures from monkey and rat aortas were different from those from human cells. In monkey, high expression of PDE1B was found, whereas PDE1C was not detected. In rat SMCs, PDE1A was the only detectable calmodulin-stimulated PDE. These findings suggest that many of the commonly used animal species may not provide good models for studying the roles of PDEs in proliferation of human SMCs. More importantly, the observation that PDE1C is induced only in proliferating SMCs suggests that it may be both an indicator of proliferation and a possible target for treatment of atherosclerosis or restenosis after angioplasty, conditions in which proliferation of arterial SMCs is negatively modulated by cyclic nucleotides.

Multiple Affinity States of cGMP-Specific Phosphodiesterase for Sildenafil Inhibition Defined by cGMP-Dependent and cGMP-Independent Mechanisms

cGMP-specific phosphodiesterase (PDE5) has become a target for drug development for the treatment of a number of physiological dysfunctions, affected by changes in the cGMP/cGMP-dependent protein kinase (PKG) signaling pathway. PDE5 has two highly homologous regulatory domains, GAF-A and GAF-B. We showed previously that PDE5 could be converted from a low-activity (nonactivated) state to a high-activity state upon cGMP binding to the GAF-A domain with higher sensitivities toward sildenafil (EMBO J 22:469–478, 2003). Here we investigated whether sildenafil sensitivity of PDE5 could be modified by cGMP-independent mechanisms. Individually expressed recombinant GAF-A and GAF-B proteins were tested for their ability to modulate full-length recombinant PDE5 affinity to sildenafil. The GAF-A domain protein had the most dramatic effect on the affinity of the nonactivated recombinant PDE5 for sildenafil, revealing much higher sensitivity to sildenafil inhibition. The apparent affinity for sildenafil increased from the nanomolar range to the picomolar range, providing evidence for the presence of a “super-high” sensitivity state of PDE5 for sildenafil inhibition. In human platelet, higher sensitivity of PDE5 for sildenafil inhibition has been detected after blocking cGMP-binding sites of the GAF-A domain. Thus, our data demonstrate that high sensitivity of PDE5 for sildenafil can be obtained not only through cGMP-induced activation of PDE, but also through cGMP-independent modulation of PDE5 in the nonactivated state, possibly through protein-protein interaction. Furthermore, data suggest that nonactivated PDE5 with “super-high” affinities for sildenafil inhibition may be responsible for therapeutic effects of long-term treatments with low doses of PDE5 inhibitors.

Differential effects of PDE5 inhibitors on cardiac dysfunction in the MDX ouse model of Duchenne muscular dystrophy

Background Duchenne muscular dystrophy (DMD) is the most common inherited form of muscular dystrophy, which results in skeletal muscle weakness by age 6. In its later stages DMD leads to dilated cardiomyopathy and heart failure with high level of mortality. At present there are no effective treatments for most of the cardiac pathology in DMD patients. Results

Calcium/Calpain mediated regulation of cyclic nucleotide phosphodiesterases, PDE3A and PDE5, in human platelets

Background Calpain, intracellular non-lysosomal calcium-dependent proteinase, is activated by a number of signaling pathways that lead to the elevation of intraplatelet calcium concentrations. Physiological roles of calpain, highly expressed in platelets, include regulation of platelet aggregation and adhesion, while excessive activation of calpain in such pathological disorders as diabetes could cause changes in platelet functions, resulting in platelet hyperactivation. Platelets from diabetic patients are also characterized by resistance to nitric oxide inhibition. Here we examined if calpain could regulate major cAMP and cGMP hydrolyzing phosphodiesterases (PDEs), PDE3A and PDE5, in human platelets.

Phosphorylation of cAMP hydrolyzing PDE (PDE3A) by cGMP-dependent protein kinase (PKG) in human platelets

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